The Overall Objectives remain to understand and define the cellular mechanisms contributing to the development and progression of biliary tract cancer, termed cholangiocarcinoma (CCA). Paradoxically, CCA cells in vivo express the death ligand TRAIL (tumor necrosis factor related apoptosis inducing ligand) and its cognate death receptors. Based on this observation, we postulate that these cancers depend upon potent survival signals to circumvent cell death by TRAIL. Consistent with this concept, we have generated preliminary data demonstrating that the Hedgehog signaling pathway upregulates X-linked inhibitor of apoptosis (XIAP) endowing CCA cells with anti-TRAIL survival signals. These cancers arise within inflammatory/fibrotic biliary tract disorders, and retain this phenotype as desmoplastic cancers. These observations indicate a critical relationship between the malignant epithelial cells and myofibroblasts within the tumor microenvironment. We have also generated striking preliminary data demonstrating that myofibroblast- derived platelet derived growth factor-BB (PDGF-BB) augments hedgehog signaling in CCA cells. Based on this preliminary data, we propose the CENTRAL HYPOTHESIS that hedgehog signaling via autocrine and myofibroblast dependent paracrine mechanisms circumvents TRAIL cytotoxicity in CCA cells. We will now employ current and complementary, molecular, biochemical and cell biological approaches to examine this hypothesis. Our SPECIFIC AIMS will test three hypotheses. FIRST, we will test the hypothesis that hedgehog inhibition sensitizes CCA cells to TRAIL-mediated cell death: a) by downregulating expression of XIAP; b) by a mitochondrial-independent pathway. SECOND, we will test the hypothesis that paracrine myofibroblast to CCA cell signaling provides anti-TRAIL survival signals: a) by myofibroblast-derived PDGF-BB which promotes hedgehog survival signaling in cholangiocarcinoma cells; and b) by myofibroblast-derived PDGF-BB-mediated, protein kinase A-dependent smoothened (the active signaling component of the Hh pathway) activation and trafficking to the plasma membrane. FINALLY, we will test the hypothesis that hedgehog and/or PDGF signaling inhibition is therapeutic in a rodent model of CCA: a) by mechanisms dependent upon tumor cell death; and b) by resulting in tumor regression and/or eradication with improved animal survival. To address these hypotheses, we have become adept at hedgehog signaling, myofibroblast:cholangiocarcinoma cell interactions, and, in collaboration, implemented a syngeneic, orthotopic, rodent model of this cancer. The proposal is technically and conceptually innovative as it identifies new mechanisms for cholangiocarcinoma cell survival signaling using a variety of sophisticated technologies. Our results will yield new mechanistic insights into CCA development and progression, and help identify strategies for the treatment and chemoprevention of CCA. PUBLIC HEALTH RELEVANCE: This application examines the cellular mechanisms by which bile duct cancer cells evade cell death. We propose that neighboring adjacent, cells, termed myofibroblasts, transmit signals to the cancer cells which enhances cancer cell generation of survival signals. We will ascertain if disruption of these survival signals results in tumor regression. The results of these studies are germane to cancer biology and have the potential to identify new therapeutic strategies for the treatment of bile duct cancer.